1. Field of the Invention
The present invention relates to methods of processing oocytes to obtain recipient oocytes for cloning, and to methods of cloning.
2. Description of the Related Art
The livestock industry in the United States is a $484 billion industry, with 99 million head of cattle producing 19 billion pounds of beef and 10 million dairy cows producing 136 billion pounds of fluid mild (USDA, 1990). Techiques exist to allow a producer to select and mate superior bulls and cows for specific traits in superior offspring. Once such a superior offspring is obtained, it is desired to produce identical animals or "clones".
Early cloning techniques included surgical bisection of an early stage embryo, prior to it losing totipotency, into halves or quarters. While these early surgical bisection techniques did increase the number of identical animals produced, these techniques were limited to 2 to 4 identicals.
Later cloning developments have allowed more advanced embryos (8-32 cells) to be manipulated into retaining totipotency of each blastomere through nuclear transplantation to oocytes, thus increasing the number of identicals up to the range of 8 to 32 identicals.
With current state of the art, mammalian offspring can be produced by nuclear transfer, which offers the potential of a much larger or (theoretically) an unlimited number of identicals. The proportion of manipulated nuclear transplant embryos developing to offspring is approximately 1% in pigs (Prather R. S., M. M. Simms, and N. L. First. 1989. Nuclear transplantation in early pig embryos. Biol. Reprod. 41:414, herein incorporated by reference), 1% (Prather R. S., F. L. Barnes, M. M. Simms, J. M. Rob, W. H. Eyestone, and First N. L. 1987. Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte. Biol. Reprod. 37:859, herein incorporated by reference) and 4% (Bondioli, K. R., M. E. Westhusin and C. R. Loony. 1990. Production of identical bovine offspring by nuclear transfer. Theriogenology 33:165, herein incorporated by reference) in cattle, approximately 4% in sheep (Smith L. C. and I. Wilmut. 1989. Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. Biol. Reprod. 40:1027, herein incorporated by reference) and 4% in rabbits (Stice S. L., and J. M. Robl. 1988. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol. Reprod. 39:657, herein incorporated by reference). A portion of the decreased developmental potential of nuclear transferred oocytes may have been due to polyploidy, as indicated by the reported enucleation rates. The enucleation rates varied from 60% in cattle (Prather et al., 1987) to 92% in rabbits (Stice and Robl, 1988). Sheep (Smith and Wilmut, 1989) and pigs (Prather et al., 1989) had rates of 68% and 74%, respectively.
While these nuclear transfer techniques for cloning identical embryos exist, they are rather costly and inefficient. With such techniques, oocytes are harvested from donor animals, with the metaphase plate subsequently removed to produce recipient oocytes. This requires a skilled technician who must remove the metaphase plate utilizing micromanipulators, and then deposit the donor blastomere through the zona pellucide into the recipient oocyte perivitelline cavity. After blastomere insertion the blastomere and recipient are fused, for example, with electrical current, chemical agent(s) or viral agent(s), and placed into development.
Removing the metaphase plate using micromanipulators is extremely time consuming, with a skilled technician capable of removing from about 100 to 200 in a 4 to 6 hour period.
Thus, there is a need in the art for an improved method of preparing recipient oocytes.
There is another need in the art for a method of preparing recipient oocytes which is less time consuming than the current methods.
There is still another need in the art for an improved method of cloning.
These and other needs in the art will become apparent to those of skill in the art upon review of this specification, including its drawings and claims.